Anti-CD3 Antibodies and Methods of Use Thereof

ABSTRACT

The present invention is related to antibodies directed to the antigen CD3 and uses of such antibodies. In particular, the present invention provides fully human monoclonal antibodies directed to CD3. Nucleotide sequences encoding, and amino acid sequences comprising, heavy and light chain immunoglobulin molecules, particularly sequences corresponding to contiguous heavy and light chain sequences spanning the framework regions and/or complementarity determining regions (CDR&#39;s), specifically from FR1 through FR4 or CDR1 through CDR3, are provided. Hybridomas or other cell lines expressing such immunoglobulin molecules and monoclonal antibodies are also provided.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/145,131, filed Jun. 3, 2005, which claims the benefit of U.S.Provisional Application No. 60/576,483, filed Jun. 3, 2004 and U.S.Provisional Application No. 60/609,153, filed Sep. 10, 2004, each ofwhich is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to fully human anti-CD3 antibodies aswell as to methods for use thereof.

BACKGROUND OF THE INVENTION

The body's immune system serves as a defense against a variety ofconditions, including, e.g., injury, infection and neoplasia, and ismediated by two separate but interrelated systems, the cellular andhumoral immune systems. Generally speaking, the humoral system ismediated by soluble products, termed antibodies or immunoglobulins,which have the ability to combine with and neutralize productsrecognized by the system as being foreign to the body. In contrast, thecellular immune system involves the mobilization of certain cells,termed T-cells that serve a variety of therapeutic roles.

The immune system of both humans and animals include two principalclasses of lymphocytes: the thymus derived cells (T cells), and the bonemarrow derived cells (B cells). Mature T cells emerge from the thymusand circulate between the tissues, lymphatics, and the bloodstream. Tcells exhibit immunological specificity and are directly involved incell-mediated immune responses (such as graft rejection). T cells actagainst or in response to a variety of foreign structures (antigens). Inmany instances these foreign antigens are expressed on host cells as aresult of infection. However, foreign antigens can also come from thehost having been altered by neoplasia or infection. Although T cells donot themselves secrete antibodies, they are usually required forantibody secretion by the second class of lymphocytes, B cells.

There are various subsets of T cells, which are generally defined byantigenic determinants found on their cell surfaces, as well asfunctional activity and foreign antigen recognition. Some subsets of Tcells, such as CD8′ cells, are killer/suppressor cells that play aregulating function in the immune system, while others, such as CD4′cells, serve to promote inflammatory and humoral responses.

Human peripheral T lymphocytes can be stimulated to undergo mitosis by avariety of agents including foreign antigens, monoclonal antibodies andlectins such as phytohemayglutinin and concanavalin A. Althoughactivation presumably occurs by binding of the mitogens to specificsites on cell membranes, the nature of these receptors, and theirmechanism of activation, is not completely elucidated. Induction ofproliferation is only one indication of T cell activation. Otherindications of activation, defined as alterations in the basal orresting state of the cell, include increased lymphokine production andcytotoxic cell activity.

T cell activation is a complex phenomenon that depends on theparticipation of a variety of cell surface molecules expressed on theresponding T cell population. For example, the antigen-specific T cellreceptor (TcR) is composed of a disulfide-linked heterodimer, containingtwo clonally distributed, integral membrane glycoprotein chains, alphaand beta (γ and δ), or gamma and delta (γ and δ), non-covalentlyassociated with a complex of low molecular weight invariant proteins,commonly designated as CD3 (once referred to as T3).

The TcR alpha and beta chains determine antigen specificities. The CD3structures represent accessory molecules that are the transducingelements of activation signals initiated upon binding of the TcR alphabeta (TcR αβ) to its ligand. There are both constant regions of theglycoprotein chains of TcR, and variable regions (polymorphisms).Polymorphic TcR variable regions define subsets of T cells, withdistinct specificities. Unlike antibodies that recognize whole orsmaller fragments of foreign proteins as antigens, the TcR complexinteracts with only small peptides of the antigen, which must bepresented in the context of major histocompatibility complex (MHC)molecules. These MHC proteins represent another highly polymorphic setof molecules randomly dispersed throughout the species. Thus, activationusually requires the tripartite interaction of the TcR and foreignpeptidic antigen bound to the major MHC proteins.

SUMMARY OF THE INVENTION

The present invention provides fully human monoclonal antibodiesspecifically directed against CD3. Exemplary monoclonal antibodiesinclude 28F11, 27H5, 23F10 and 15C3 described herein. Alternatively, themonoclonal antibody is an antibody that binds to the same epitope as28F11, 27H5, 23F10 or 15C3. The antibodies are respectively referred toherein is huCD3 antibodies. The huCD3 antibody has one or more of thefollowing characteristics: the antibody binds to CD3 positive (CD3+)cells but not CD3 negative (CD3−) cells; the huCD3 antibody inducesantigenic modulation which involves alteration (e.g., decrease) of thecell surface expression level or activity of CD3 or the T cell receptor(TcR); the huCD3 antibody inhibits binding of the murine anti-human OKT3monoclonal antibody to T-lymphocytes; or the huCD3 antibody binds anepitope of CD3 that wholly or partially includes the amino acid sequenceEMGGITQTPYKVSISGT (SEQ ID NO:67). The huCD3 antibodies of the inventioncompete with the murine anti-CD3 antibody OKT3 for binding to CD3, andexposure to the huCD3 antibody removes or masks CD3 and/or TcR withoutaffecting cell surface expression of CD2, CD4 or CD8. The masking of CD3and/or TcR results in the loss or reduction of T-cell activation, whichis desirable in autoimmune diseases where uncontrolled T-cell activationoccurs. Down-regulation of CD3 results in a prolonged effect of reducedT cell activation, e.g., for a period of at least several months, ascompared with the transient suppression that is observed when using atraditional immunosuppressive agent, e.g., cyclosporin.

Antigenic modulation refers to the redistribution and elimination of theCD3-T cell receptor complex on the surface of a cell, e.g., alymphocyte. Decrease in the level of cell surface expression or activityof the TcR on the cell is meant that the amount or function of the TcRis reduced. Modulation of the level of cell surface expression oractivity of CD3 is meant that the amount of CD3 on the cell surface orfunction of CD3 is altered, e.g., reduced. The amount of CD3 or the TcRexpressed at the plasma membrane of the cell is reduced, for example, byinternalization of CD3 or the TcR upon contact of the cell with thehuCD3 antibody. Alternatively, upon contact of a cell with the huCD3antibody, CD3 or the TcR is masked.

Inhibiting the binding of the murine anti-human OKT3 monoclonal antibodyto a T-lymphocyte is defined as a decrease in the ability of the murineOKT3 antibody to form a complex with CD3 on the cell surface of aT-lymphocyte.

A huCD3 antibody contains a heavy chain variable having the amino acidsequence of SEQ ID NOS: 2, 6, 10 or 22 and a light chain variable havingthe amino acid sequence of SEQ ID NOS: 4, 8, 16-20 or 25-26. Preferably,the three heavy chain CDRs include an amino acid sequence at least 90%,92%, 95%, 97% 98%, 99% or more identical a sequence selected from thegroup consisting of GYGMH (SEQ ID NO:27); VIWYDGSKKYYVDSVKG (SEQ IDNO:28); QMGYWHFDL (SEQ ID NO:29); SYGMH (SEQ ID NO:33);IIWYDGSKKNYADSVKG (SEQ ID NO:34); GTGYNWFDP (SEQ ID NO:35); andAIWYNGRKQDYADSVKG (SEQ ID NO:44) and a light chain with three CDR thatinclude an amino acid sequence at least 90%, 92%, 95%, 97% 98%, 99% ormore identical to a sequence selected from the group consisting of theamino acid sequence of RASQSVSSYLA (SEQ ID NO:30); DASNRAT (SEQ IDNO:31); QQRSNWPPLT (SEQ ID NO:32); RASQSVSSSYLA (SEQ ID NO:36); GASSRAT(SEQ ID NO:37); QQYGSSPIT (SEQ ID NO:38); RASQGISSALA (SEQ ID NO:39);YASSLQS (SEQ ID NO:40); QQYYSTLT (SEQ ID NO:41); DASSLGS (SEQ ID NO:42);WASQGISSYLA (SEQ ID NO:43); QQRSNWPWT (SEQ ID NO:45); DASSLES (SEQ IDNO:46); and QQFNSYPIT (SEQ ID NO:47). The antibody binds CD3.

A huCD3 antibody of the invention exhibits at least two or more (i.e.,two or more, three or more, four or more, five or more, six or more,seven or more, eight or more, nine or more, ten or more, eleven or more)of the following characteristics: the antibody contains a variable heavychain region (V_(H)) encoded by a human DP50 V_(H) germline genesequence, or a nucleic acid sequence that is homologous to the humanDP50 V_(H) germline gene sequence; the antibody contains a variablelight chain region (V_(L)) encoded by a human L6 V_(L) germline genesequence, or a nucleic acid sequence homologous to the human L6 V_(L)germline gene sequence; the antibody contains a V_(L) encoded by a humanL4/18a V_(L) germline gene sequence, or a nucleic acid sequencehomologous to the human L4/18a V_(L) germline gene sequence; theantibody includes a V_(H) CDR1 region comprising the amino acid sequenceYGMH (SEQ ID NO:58); the antibody includes a V_(H) CDR2 regioncomprising the amino acid sequence DSVKG (SEQ ID NO:59); the antibodyincludes a V_(H) CDR2 region comprises the amino acid sequenceIWYX₁GX₂X₃X₄X₅Y X₆DSVKG (SEQ ID NO:60); the antibody includes a V_(H)CDR3 region comprising the amino acid sequenceX_(A)X_(B)GYX_(C)X_(D)FDX_(E) (SEQ ID NO:61); the antibody includes aV_(H) CDR3 region comprising the amino acid sequence GTGYNWFDP (SEQ IDNO:62) or the amino acid sequence QMGYWHFDL (SEQ ID NO:63); the antibodyincludes the amino acid sequence VTVSS (SEQ ID NO:64) at a position thatis C-terminal to the CDR3 region, wherein the position is in a variableregion C-terminal to the CDR3 region; the antibody includes the aminoacid sequence GTLVTVSS (SEQ ID NO:65) at a position that is C-terminalto CDR3 region, wherein the position is in a variable region C-terminalto the CDR3 region; the antibody includes the amino acid sequenceWGRGTLVTVSS (SEQ ID NO:66) at a position that is C-terminal to CDR3region, wherein the position is in a variable region C-terminal to theCDR3 region; the antibody binds an epitope that wholly or partiallyincludes the amino acid sequence EMGGITQTPYKVSISGT (SEQ ID NO:67); andthe antibody includes a mutation in the heavy chain at an amino acidresidue at position 234, 235, 265, or 297 or combinations thereof, andwherein the release of cytokines from a T-cell in the presence of saidantibody is reduced as compared to the release of cytokines from aT-cell in the presence of an antibody that does not include a mutationin the heavy chain at position 234, 235, 265 or 297 or combinationsthereof. The numbering of the heavy chain residues described herein isthat of the EU index (see Kabat et al., “Proteins of ImmunologicalInterest”, US Dept. of Health & Human Services (1983)), as shown, e.g.,in U.S. Pat. Nos. 5,624,821 and 5,648,260, the contents of which arehereby incorporated in its entirety by reference.

In some aspects, the huCD3 antibody contains an amino acid mutation. Themutation is in the constant region. The mutation results in an antibodythat has an altered effector function. An effector function of anantibody is altered by altering, i.e., enhancing or reducing, theaffinity of the antibody for an effector molecule such as an Fc receptoror a complement component. By altering an effector function of anantibody, it is possible to control various aspects of the immuneresponse, e.g., enhancing or suppressing various reactions of the immunesystem. For example, the mutation results in an antibody that is capableof reducing cytokine release from a T-cell. For example, the mutation isin the heavy chain at amino acid residue 234, 235, 265, or 297 orcombinations thereof. Preferably, the mutation results in an alanineresidue at either position 234, 235, 265 or 297, or a glutamate residueat position 235, or a combination thereof. The term “cytokine” refers toall human cytokines known within the art that bind extracellularreceptors expressed on the cell surface and thereby modulate cellfunction, including but not limited to IL-2, IFN-gamma, TNF-a, IL-4,IL-5, IL-6, IL-9, IL-10, and IL-13.

The release of cytokines can lead to a toxic condition known as cytokinerelease syndrome (CRS), a common clinical complication that occurs,e.g., with the use of an anti-T cell antibody such as ATG(anti-thymocyte globulin) and OKT3 (a murine anti-human CD3 antibody).This syndrome is characterized by the excessive release of cytokinessuch as TNF, IFN-gamma and IL-2 into the circulation. The CRS occurs asa result of the simultaneous binding of the antibodies to CD3 (via thevariable region of the antibody) and the Fc Receptors and/or complementreceptors (via the constant region of the antibody) on other cells,thereby activating the T cells to release cytokines that produce asystemic inflammatory response characterized by hypotension, pyrexia andrigors. Symptoms of the CRS include fever, chills, nausea, vomiting,hypotension, and dyspnea. Thus, the huCD3 antibody of the inventioncontains one or more mutations that prevent heavy chain constantregion-mediated release of one or more cytokine(s) in vivo.

The fully human CD3 antibodies of the invention include, for example, aL²³⁴ L²³⁵→A²³⁴ E²³⁵ mutation in the Fc region, such that cytokinerelease upon exposure to the huCD3 antibody is significantly reduced oreliminated (see e.g., FIGS. 11A, 11B). As described below in Example 4,the L²³⁴ L²³⁵→A²³⁴ E²³⁵ mutation in the Fc region of the huCD3antibodies of the invention reduces or eliminates cytokine release whenthe huCD3 antibodies are exposed to human leukocytes, whereas themutations described below maintain significant cytokine releasecapacity. For example, a significant reduction in cytokine release isdefined by comparing the release of cytokines upon exposure to the huCD3antibody having a L²³⁴ L²³⁵→A²³⁴ E²³⁵ mutation in the Fc region to levelof cytokine release upon exposure to another anti-CD3 antibody havingone or more of the mutations described below. Other mutations in the Fcregion include, for example, L²³⁴ L²³⁵→A²³⁴ A²³⁵, L²³⁵→E²³⁵, N²⁹⁷→A²⁹⁷,and D²⁶⁵→A²⁶⁵.

Alternatively, the huCD3 antibody is encoded by a nucleic acid thatincludes one or more mutations that replace a nucleic acid residue witha germline nucleic acid residue. By “germline nucleic acid residue” ismeant the nucleic acid residue that naturally occurs in a germline geneencoding a constant or variable region. “Germline gene” is the DNA foundin a germ cell (i.e., a cell destined to become an egg or in the sperm).A “germline mutation” refers to a heritable change in a particular DNAthat has occurred in a germ cell or the zygote at the single-cell stage,and when transmitted to offspring, such a mutation is incorporated inevery cell of the body. A germline mutation is in contrast to a somaticmutation which is acquired in a single body cell. In some cases,nucleotides in a germline DNA sequence encoding for a variable regionare mutated (i.e., a somatic mutation) and replaced with a differentnucleotide. Thus, the antibodies of the invention include one or moremutations that replace a nucleic acid with the germline nucleic acidresidue. Germline antibody genes include, for example, DP50 (Accessionnumber: IMGT/EMBL/GenBank/DDBJ: L06618), L6 (Accession number:IMGT/EMBL/GenBank/DDBJ: X01668) and L4/18a (Accession number:EMBL/GenBank/DDBJ: Z00006).

The heavy chain of a huCD3 antibody is derived from a germ line V(variable) gene such as, for example, the DP50 germline gene. Thenucleic acid and amino acid sequences for the DP50 germline geneinclude, for example, the nucleic acid and amino acid sequences shownbelow:

(SEQ ID NO: 68) tgattcatgg agaaatagag agactgagtg tgagtgaaca tgagtgagaaaaactggatt tgtgtggcat tttctgataa cggtgtcctt ctgtttgcag gtgtccagtgtcaggtgcag ctggtggagt ctgggggagg cgtggtccag cctgggaggt ccctgagactctcctgtgca gcgtctggat tcaccttcag tagctatggc atgcactggg tccgccaggctccaggcaag gggctggagt gggtggcagt tatatggtat gatggaagta ataaatactatgcagactcc gtgaagggcc gattcaccat ctccagagac aattccaaga acacgctgtatctgcaaatg aacagcctga gagccgagga cacggctgtg tattactgtg cgagagacac ag(SEQ ID NO: 69) VQCQVQLVES GGGVVQPGRS LRLSCAASGF TFSSYGMHWV RQAPGKGLEWVAVIWYDGSN KYYADSVKGR FTISRDNSKN TLYLQMNSLR AEDTAVYYCA R

The huCD3 antibodies of the invention include a variable heavy chain(V_(H)) region encoded by a human DP50 V_(H) germline gene sequence. ADP50 V_(H) germline gene sequence is shown, e.g., in SEQ ID NO:48 inFIG. 5. The huCD3 antibodies of the invention include a V_(H) regionthat is encoded by a nucleic acid sequence that is at least 80%homologous to the DP50 V_(H) germline gene sequence. Preferably, thenucleic acid sequence is at least 90%, 95%, 96%, 97% homologous to theDP50 V_(H) germline gene sequence, and more preferably, at least 98%,99% homologous to the DP50 V_(H) germline gene sequence. The V_(H)region of the huCD3 antibody is at least 80% homologous to the aminoacid sequence of the V_(H) region encoded by the DP50 V_(H) germlinegene sequence. Preferably, the amino acid sequence of V_(H) region ofthe huCD3 antibody is at least 90%, 95%, 96%, 97% homologous to theamino acid sequence encoded by the DP50 V_(H) germline gene sequence,and more preferably, at least 98%, 99% homologous to the sequenceencoded by the DP50 V_(H) germline gene sequence.

The huCD3 antibodies of the invention also include a variable lightchain (V_(L)) region encoded by a human L6 or L4/18a V_(L) germline genesequence. A human L6 V_(L) germline gene sequence is shown, e.g., in SEQID NO:70 in FIG. 6, and a human L4/18a V_(L) germline gene sequence isshown, for example, in SEQ ID NO:53 in FIG. 7. Alternatively, the huCD3antibodies include a V_(L) region that is encoded by a nucleic acidsequence that is at least 80% homologous to either the L6 or L4/18aV_(L) germline gene sequence. Preferably, the nucleic acid sequence isat least 90%, 95%, 96%, 97% homologous to either the L6 or L4/18a V_(L)germline gene sequence, and more preferably, at least 98%, 99%homologous to either the L6 or L4/18a V_(L) germline gene sequence. TheV_(L) region of the huCD3 antibody is at least 80% homologous to theamino acid sequence of the V_(L) region encoded by either the L6 orL4/18a V_(L) germline gene sequence. Preferably, the amino acid sequenceof V_(L) region of the huCD3 antibody is at least 90%, 95%, 96%, 97%homologous to the amino acid sequence encoded by either the L6 or L4/18aV_(L) germline gene sequence, and more preferably, at least 98%, 99%homologous to the sequence encoded by either the L6 or L4/18a V_(L)germline gene sequence.

The huCD3 antibodies of the invention have, for example, partiallyconserved amino acid sequences that are derived from the DP50 germline.For example, the CDR1 region of huCD3 antibodies of the invention haveat least the contiguous amino acid sequence YGMH (SEQ ID NO: 58).

The CDR2 of the huCD3 antibodies includes, e.g., at least the contiguousamino acid sequence DSVKG (SEQ ID NO:59). For example, the CDR2 regionincludes the contiguous amino acid sequence IWYX₁GX₂X₃X₄X₅YX₆DSVKG (SEQID NO:60), where X₁, X₂, X₃, X₄, X₅ and X₆ represent any amino acid. Forexample, X_(i), X₂, X₃ and X₄ are hydrophilic amino acids. In some huCD3antibodies of the invention, X_(i) is asparagine or aspartate, X₂ isarginine or serine, X₃ is lysine or asparagine, X₄ is lysine orglutamine, X₅ is aspartate, asparagine or tyrosine, and/or X₆ is valineor alanine. For example, the V_(H) CDR2 region includes an amino acidsequence selected from the group consisting of AIWYNGRKQDYADSVKG (SEQ IDNO:44), IIWYDGSKKNYADSVKG (SEQ ID NO:34), VIWYDGSKKYYVDSVKG (SEQ IDNO:28) and VIWYDGSNKYYADSVKG (SEQ ID NO:72).

The CDR3 region of huCD3 antibodies contain, for example, at least thecontiguous amino acid sequence X_(A)X_(B)GYX_(C)X_(D)FDX_(E) (SEQ IDNO:61), where X_(A), X_(B), X_(C), X_(D), and X_(E) represent any aminoacid. In some huCD3 antibodies of the invention, X_(A) and X_(B) areneutral amino acids, X_(D) is an aromatic amino acid, and/or whereinX_(E) is a hydrophobic amino acid. For example, X_(A) is glycine orglutamine, X_(B) is threonine or methionine, X_(C) is asparagine ortryptophan, X_(D) is tryptophan or histidine, and/or X_(E) is proline orleucine. For example, the CDR3 region includes either the contiguousamino acid sequence GTGYNWFDP (SEQ ID NO:62) or the contiguous aminoacid sequence QMGYWHFDL (SEQ ID NO: 63).

The huCD3 antibodies include a framework 2 region (FRW2) that containsthe amino acid sequence WVRQAPGKGLEWV (SEQ ID NO:73). huCD3 antibodiesof the invention include a framework 3 region (FRW3) that contains theamino acid sequence RFTISRDNSKNTLYLQMNSLRAEDTAVYYCA (SEQ ID NO:74).

Some huCD3 antibodies include the contiguous amino acid sequence VTVSS(SEQ ID NO:64) at a position that is C-terminal to CDR3 region. Forexample, the antibody contains the contiguous amino acid sequenceGTLVTVSS (SEQ ID NO:65) at a position that is C-terminal to the CDR3region. Other huCD3 antibodies include the contiguous amino acidsequence WGRGTLVTVSS (SEQ ID NO: 66) at a position that is C-terminal tothe CDR3 region. The arginine residue in SEQ ID NO:66 is shown, forexample, in the V_(H) sequences for the 28F11 huCD3 antibody (SEQ IDNO:2) and the 23F10 huCD3 antibody (SEQ ID NO:6).

In another aspect, the invention provides methods of treating,preventing or alleviating a symptom of an immune-related disorder byadministering an huCD3 antibody to a subject. Optionally, the subject isfurther administered with a second agent such as, but not limited to,anti-inflammatory compounds or immunosuppressive compounds. For example,subjects with Type I diabetes or Latent Autoimmune Diabetes in the Adult(LADA), are also administered a second agent, such as, for example,GLP-1 or a beta cell resting compound (i.e., a compound that reduces orotherwise inhibits insulin release, such as potassium channel openers).

Suitable compounds include, but are not limited to methotrexate,cyclosporin A (including, for example, cyclosporin microemulsion),tacrolimus, corticosteroids, statins, interferon beta, Remicade(Infliximab), Enbrel (Etanercept) and Humira (Adalimumab).

The subject is suffering from or is predisposed to developing an immunerelated disorder, such as, for example, an autoimmune disease or aninflammatory disorder.

In another aspect, the invention provides methods of administering thehuCD3 antibody of the invention to a subject prior to, during and/orafter organ or tissue transplantation. For example, the huCD3 antibodyof the invention is used to treat or prevent rejection after organ ortissue transplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of representations of the nucleotide sequence andamino acid sequences for the variable light and variable heavy regionsof the huCD3 antibody 28F11. FIG. 1A depicts the nucleotide sequenceencoding the variable region of the heavy chain, and FIG. 1B representsthe amino acid sequence encoded by the nucleotide sequence shown in FIG.1A, wherein the CDRs are highlighted with boxes. FIG. 1C depicts thenucleotide sequence encoding the variable region of the light chain, andFIG. 1D represents the amino acid sequence encoded by the nucleotidesequence shown in FIG. 1C, wherein the CDRs are indicated by boxes.

FIG. 2 is series of representations of the nucleotide sequence and aminoacid sequences for the variable light and variable heavy regions of thehuCD3 antibody 23F10, with FIG. 2A representing the nucleotide sequenceencoding the variable region of the heavy chain, FIG. 2B representingthe amino acid sequence encoded by the nucleotide sequence shown in FIG.2A, FIG. 2C representing the nucleotide sequence encoding the variableregion of the light chain, and FIG. 2D representing the amino acidsequence encoded by the nucleotide sequence shown in FIG. 2C.

FIG. 3 is series of representations of the nucleotide sequence and aminoacid sequences for the variable light and variable heavy regions of thehuCD3 antibody 27H5. FIG. 3A represents the nucleotide sequence encodingthe variable region of the heavy chain; FIG. 3B represents the aminoacid sequence encoded by the nucleotide sequence shown in FIG. 3A; FIG.3C represents the five nucleotide sequences encoding the variable regionof the light chain for the 27H5 clone; FIG. 3D represents the five aminoacid sequences encoded by the nucleotide sequences shown in FIG. 3C; andFIG. 3E is an alignment of the five light chains from the clone 27H5,wherein an asterisk (*) in the last row (labeled KEY) represents aconserved amino acid in that column; a colon (:) in the KEY rowrepresents a conservative mutation; and a period (.) in the KEY rowrepresents a semiconservative mutation.

FIG. 4 is a is series of representations of the nucleotide sequence andamino acid sequences for the variable light and variable heavy regionsof the huCD3 antibody 15C3, with FIG. 4A representing the nucleotidesequence encoding the variable region of the heavy chain, FIG. 4Brepresenting the amino acid sequence encoded by the nucleotide sequenceshown in FIG. 4A, FIG. 4C representing the two nucleotide sequencesencoding the variable region of the light chain for the 15C3 clone, andFIG. 4D representing the two amino acid sequences encoded by thenucleotide sequences shown in FIG. 4C.

FIG. 5 is an alignment depicting the variable heavy chain regions of the15C3, 27H5 and 28F11 huCD3 antibodies as well as the DP-50 germlinesequence, the human heavy joining 5-02 sequence, and the human heavyjoining 2 sequence. The CDR regions are indicated for each sequence.

FIG. 6 is an alignment depicting the VκIII variable regions of the 15C3(variable light chain 1, i.e., “VL1”) and 28F11 huCD3 antibodies, aswell as the L6 germline sequence, the human kappa joining 4 sequence andthe human kappa joining 1 sequence. The CDR regions are indicated foreach sequence.

FIG. 7 is an alignment depicting the VκI variable regions of the 15C3(variable light chain 2, i.e., “VL2”) and 27H5 VL2 huCD3 antibodies, aswell as the L4/18a germline sequence, the human kappa joining 4 sequenceand the human kappa joining 5 sequence. The CDR regions are indicatedfor each sequence.

FIG. 8 is an alignment depicting the VκII variable regions of the 27H5VL1 huCD3 antibody and DPK22, as well as human kappa joining 5 sequence.The CDR regions are indicated for each sequence.

FIG. 9A is a graph depicting antibody binding to CD3 molecules at thesurface of Jurkat cells using a variety of anti-CD3 antibodies,including the 28F11, 27H5VL1, 27H5VL2, 15C3VL1 and 15C3VL2 huCD3antibodies of the invention. FIG. 9B is a graph depicting the ability ofa variety of anti-CD3 antibodies, including the 28F11, 27H5VL1, 27H5VL2,15C3VL1 and 15C3VL2 huCD3 antibodies of the invention, to inhibit thebinding of the murine anti-CD3 antibody OKT3 to CD3 positive cells. FIG.9C is a graph depicting the antigenic modulation of CD3 and TCR from thesurface of human peripheral blood T cells by a variety of anti-CD3antibodies, including the 28F11, 27H5VL1, 27H5VL2, 15C3VL1 and 15C3VL2huCD3 antibodies of the invention. FIG. 9D is a graph depicting theeffect of a variety anti-CD3 antibodies, including the 28F11, 27H5VL1,27H5VL2, 15C3VL1 and 15C3VL2 huCD3 antibodies of the invention, onT-cell proliferation.

FIG. 10 is an illustration depicting the binding pattern of the fullyhuman monoclonal antibody 28F11 on a peptide array derived from theamino acid sequence of the CD3 epsilon chain.

FIG. 11 is a series of graphs depicting the level of cytokine releaseupon exposure to wild-type 28F11 huCD3 antibody (28F11WT), a mutated28F11 huCD3 antibody having a L²³⁴ L²³⁵→A²³⁴ A²³⁵ mutation (28F11AA),and a mutated 28F11 huCD3 antibody having a L²³⁴ L²³⁵→A²³⁴ E²³⁵ mutation(28F11AE). FIG. 11A depicts the level of TNF-alpha release upon exposureto these antibodies, and FIG. 11B depicts the level of interferon gammarelease.

DETAILED DESCRIPTION

The present invention provides fully human monoclonal antibodiesspecific against CD3 epsilon chain (CDR). The antibodies arerespectively referred to herein is huCD3 antibodies.

CD3 is a complex of at least five membrane-bound polypeptides in matureT-lymphocytes that are non-covalently associated with one another andwith the T-cell receptor. The CD3 complex includes the gamma, delta,epsilon, zeta, and eta chains (also referred to as subunits). Non-humanmonoclonal antibodies have been developed against some of these chains,as exemplified by the murine antibodies OKT3, SP34, UCHT1 or 64.1. (Seee.g., Ledbetter, et al., J. Immunol. 136:3945-3952 (1986); Yang, et al.,J. Immunol. 137:1097-1100 (1986); and Hayward, et al., Immunol. 64:87-92(1988)).

The huCD3 antibodies of the invention were produced by immunizing twolines of transgenic mice, the HuMab™ mice and the KM™ mice (Medarex,Princeton N.J.).

The huCD3 antibodies of the invention have one or more of the followingcharacteristics: the huCD3 antibody binds to CD3 positive (CD3+) cellsbut not CD3 negative (CD3−) cells; the huCD3 antibody induces antigenicmodulation which involves alterations of the cell surface expressionlevels of CD3 and the T cell receptor (TcR); or the huCD3 antibodyinhibits binding of the murine anti-human OKT3 monoclonal antibody toT-lymphocytes. The huCD3 antibodies of the invention compete with themurine anti-CD3 antibody OKT3 for binding to CD3, and exposure to thehuCD3 antibody removes or masks CD3 and/or TcR without affecting cellsurface expression of CD2, CD4 or CD8. The masking of CD3 and/or TcRresults in the loss or reduction of T-cell activation.

The huCD3 antibodies of the invention bind to a CD3 that wholly orpartially includes the amino acid residues from position 27 to position43 of the processed human CD3 epsilon subunit (i.e., without the leadersequence). The amino acid sequence of the human CD3 epsilon subunit isshown, for example, in GenBank Accession Nos. NP 000724; AAA52295;P07766; A32069; CAA27516; and AAH49847. For example, the huCD3 antibodybinds a CD3 epitope that wholly or partially includes the amino acidsequence of EMGGITQTPYKVSISGT (SEQ ID NO: 67). An exemplary huCD3monoclonal antibody that binds to this epitope is the 28F11 antibodydescribed herein. The 28F11 antibody includes a heavy chain variableregion (SEQ ID NO:2) encoded by the nucleic acid sequence shown below inSEQ ID NO:1, and a light chain variable region (SEQ ID NO:4) encoded bythe nucleic acid sequence shown in SEQ ID NO:3 (FIGS. 1A-1D).

The amino acids encompassing the complementarity determining regions(CDR) as defined by Chothia et al. 1989, E. A. Kabat et al., 1991 arehighlighted with boxes below (see also FIGS. 1B and 1D and FIGS. 5 and6). (See Chothia, C, et al., Nature 342:877-883 (1989); Kabat, E A, etal., Sequences of Protein of immunological interest, Fifth Edition, USDepartment of Health and Human Services, US Government Printing Office(1991)). The heavy chain CDRs of the 28F11 antibody have the followingsequences: GYGMH (SEQ ID NO:27) VIWYDGSKKYYVDSVKG (SEQ ID NO:28) andQMGYWHFDL (SEQ ID NO:29). The light chain CDRs of the 28F11 antibodyhave the following sequences: RASQSVSSYLA (SEQ ID NO:30) DASNRAT (SEQ IDNO:31) and QQRSNWPPLT (SEQ ID NO:32).

The 23F10 antibody includes a heavy chain variable region (SEQ ID NO:6)encoded by the nucleic acid sequence shown below in SEQ ID NO:5, and alight chain variable region (SEQ ID NO:8) encoded by the nucleic acidsequence shown in SEQ ID NO:7.

The amino acids encompassing the CDR as defined by Chothia et al. 1989,E. A. Kabat et al., 1991 are highlighted with boxes below. (see alsoFIGS. 2B, 2D). The heavy chain CDRs of the 23F10 antibody have thefollowing sequences: GYGMH (SEQ ID NO:27) VIWYDGSKKYYVDSVKG (SEQ IDNO:28) and QMGYWHFDL (SEQ ID NO:29). The light chain CDRs of the 23F10antibody have the following sequences: RASQSVSSYLA (SEQ ID NO:30)DASNRAT (SEQ ID NO:31) and QQRSNWPPLT (SEQ ID NO:32).

The 27H5 antibody includes a heavy chain variable region (SEQ ID NO:10)encoded by the nucleic acid sequence shown below in SEQ ID NO:9, and alight chain variable region selected from the amino acid sequences shownbelow in SEQ ID NOS: 16-20 and encoded by the nucleic acid sequencesshown in SEQ ID NO:11-15. As described herein in Example 2, a singleclonal hybridoma derived from the HuMAb® transgenic mice can producemultiple light chains for a single heavy chain. Each combination ofheavy and light chains produced is tested for optimal functioning, asdescribed herein in Example 2.

The amino acids encompassing the CDR as defined by Chothia et al. 1989,E. A. Kabat et al., 1991 are highlighted with boxes below. (see alsoFIGS. 3B, 3D, 5, and 7-8). The heavy chain CDRs of the 27H5 antibodyhave the following sequences: SYGMH (SEQ ID NO:33) IIWYDGSKKNYADSVKG(SEQ ID NO:34) and GTGYNWFDP (SEQ ID NO:35). The light chain CDRs of the27H5 antibody have the following sequences: RASQSVSSSYLA (SEQ ID NO:36);GASSRAT (SEQ ID NO:37); QQYGSSPIT (SEQ ID NO:38); RASQGISSALA (SEQ IDNO:39); YASSLQS (SEQ ID NO:40); QQYYSTLT (SEQ ID NO:41); DASSLGS (SEQ IDNO:42); and WASQGISSYLA (SEQ ID NO:43).

The 15C3 antibody includes a heavy chain variable region (SEQ ID NO:22)encoded by the nucleic acid sequence shown below in SEQ ID NO:21, and alight chain variable region selected from the amino acid sequences shownbelow in SEQ ID NOS: 25-26 and encoded by the nucleic acid sequencesshown in SEQ ID NO:23-24. As described herein in Example 2, a singleclonal hybridoma derived from the HuMAb® transgenic mice can producemultiple light chains for a single heavy chain. Each combination ofheavy and light chains produced is tested for optimal functioning, asdescribed herein in Example 2.

The amino acids encompassing the CDR as defined by Chothia et al. 1989,E. A. Kabat et al., 1991 are highlighted with boxes below. (see alsoFIGS. 4B, 4D, and 5-7). The heavy chain CDRs of the 15C3 antibody havethe following sequences: SYGMH (SEQ ID NO:33) AIWYNGRKQDYADSVKG (SEQ IDNO:44) and GTGYNWFDP (SEQ ID NO:35). The light chain CDRs of the 15C3antibody have the following sequences: RASQSVSSYLA (SEQ ID NO:30);DASNRAT (SEQ ID NO:31); QQRSNWPWT (SEQ ID NO:45); RASQGISSALA (SEQ IDNO:39); DASSLES (SEQ ID NO:46); QQFNSYPIT (SEQ ID NO:47).

huCD3 antibodies of the invention also include antibodies that include aheavy chain variable amino acid sequence that is at least 90%, 92%, 95%,97% 98%, 99% or more identical the amino acid sequence of SEQ ID NO:2,6, 10 or 22 and/or a light chain variable amino acid that is at least90%, 92%, 95%, 97% 98%, 99% or more identical the amino acid sequence ofSEQ ID NO:4, 8, 16-20 or 25-26.

Alternatively, the monoclonal antibody is an antibody that binds to thesame epitope as 28F11, 27H5, 23F10 or 15C3.

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques are used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. Such antibodies include, but arenot limited to, polyclonal, monoclonal, chimeric, single chain, F_(ab),F_(ab′) and F_((ab′)2) fragments, and an F_(ab) expression library. By“specifically bind” or “immunoreacts with” is meant that the antibodyreacts with one or more antigenic determinants of the desired antigenand does not react (i.e., bind) with other polypeptides or binds at muchlower affinity (K_(d)>10⁻⁶) with other polypeptides.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ea., 2nd ed. Raven Press, N.Y.(1989)). The variable regions of each light/heavy chain pair form theantibody binding site.

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

In general, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “antigen-binding site,” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.” Theassignment of amino acids to each domain is in accordance with thedefinitions of Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987 and 1991)), orChothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature342:878-883 (1989).

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, an scFv, or a T-cellreceptor. The term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. An antibody is said to specificallybind an antigen when the dissociation constant is ≦1 μM; preferably ≦100nM and most preferably ≦10 nM.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides are quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to a CD3 epitope when the equilibrium bindingconstant (K_(d)) is ≦1 μM, preferably ≦100 nM, more preferably ≦10 nM,and most preferably ≦100 μM to about 1 pM, as measured by assays such asradioligand binding assays or similar assays known to those skilled inthe art.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a human monoclonal antibodyhas the same specificity as a human monoclonal antibody of the invention(e.g., monoclonal antibody 28F11, 27H5, 23F10 or 15C3) by ascertainingwhether the former prevents the latter from binding to a CD3 antigenpolypeptide. If the human monoclonal antibody being tested competes witha human monoclonal antibody of the invention, as shown by a decrease inbinding by the human monoclonal antibody of the invention, then the twomonoclonal antibodies bind to the same, or a closely related, epitope.Another way to determine whether a human monoclonal antibody has thespecificity of a human monoclonal antibody of the invention is topre-incubate the human monoclonal antibody of the invention with the CD3antigen polypeptide with which it is normally reactive, and then add thehuman monoclonal antibody being tested to determine if the humanmonoclonal antibody being tested is inhibited in its ability to bind theCD3 antigen polypeptide. If the human monoclonal antibody being testedis inhibited then, in all likelihood, it has the same, or functionallyequivalent, epitopic specificity as the monoclonal antibody of theinvention.

Various procedures known within the art are used for the production ofthe monoclonal antibodies directed against a protein such as a CD3protein, or against derivatives, fragments, analogs homologs ororthologs thereof. (See, e.g., Antibodies: A Laboratory Manual, HarlowE, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., incorporated herein by reference). Fully human antibodiesare antibody molecules in which the entire sequence of both the lightchain and the heavy chain, including the CDRs, arise from human genes.Such antibodies are termed “human antibodies”, or “fully humanantibodies” herein. Human monoclonal antibodies are prepared, forexample, using the procedures described below in Example 1. Humanmonoclonal antibodies can be also prepared by using trioma technique;the human B-cell hybridoma technique (see Kozbor, et al., 1983 ImmunolToday 4: 72); and the EBV hybridoma technique to produce humanmonoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIESAND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonalantibodies may be utilized and may be produced by using human hybridomas(see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or bytransforming human B-cells with Epstein Barr Virus in vitro (see Cole,et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,Inc., pp. 77-96).

Antibodies are purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

It is desirable to modify the antibody of the invention with respect toeffector function, so as to enhance, e.g., the effectiveness of theantibody in treating immune-related diseases. For example, cysteineresidue(s) can be introduced into the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated can have improved internalization capabilityand/or increased complement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)).Alternatively, an antibody can be engineered that has dual Fc regionsand can thereby have enhanced complement lysis and ADCC capabilities.(See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)).

The invention also includes F_(v), F_(ab), F_(ab′) and F_((ab′)2) huCD3fragments, single chain huCD3 antibodies, bispecific huCD3 antibodiesand heteroconjugate huCD3 antibodies.

Bispecific antibodies are antibodies that have binding specificities forat least two different antigens. In the present case, one of the bindingspecificities is for CD3. The second binding target is any otherantigen, and advantageously is a cell-surface protein or receptor orreceptor subunit.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO 96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. The preferred interface comprises at least a part of the CH3region of an antibody constant domain. In this method, one or more smallamino acid side chains from the interface of the first antibody moleculeare replaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniques forgenerating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Additionally, Fab′ fragments can be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

Exemplary bispecific antibodies can bind to two different epitopes, atleast one of which originates in the protein antigen of the invention.Alternatively, an anti-antigenic arm of an immunoglobulin molecule canbe combined with an arm which binds to a triggering molecule on aleukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, orB7), or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32)and FcγRIII (CD16) so as to focus cellular defense mechanisms to thecell expressing the particular antigen. Bispecific antibodies can alsobe used to direct cytotoxic agents to cells which express a particularantigen. These antibodies possess an antigen-binding arm and an armwhich binds a cytotoxic agent or a radionuclide chelator, such asEOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interestbinds the protein antigen described herein and further binds tissuefactor (TF).

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP03089). It is contemplated that the antibodies can be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins can beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies or toother molecules of the invention. (See, for example, “ConjugateVaccines”, Contributions to Microbiology and Immunology, J. M. Cruse andR. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entirecontents of which are incorporated herein by reference).

Coupling is accomplished by any chemical reaction that will bind the twomolecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding is achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987). Preferred linkers are describedin the literature. (See, for example, Ramakrishnan, S. et al., CancerRes. 44:201-208 (1984) describing use of MBS(M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat. No.5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Particularlypreferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NHS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of marine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein fragments, and analogs are species of the polypeptidegenus. Preferred polypeptides in accordance with the invention comprisethe human heavy chain immunoglobulin molecules represented by FIGS. 1B,2B, 3B and 4B and the human light chain immunoglobulin moleculesrepresented by FIGS. 1D, 2D, 3D and 4D, as well as antibody moleculesformed by combinations comprising the heavy chain immunoglobulinmolecules with light chain immunoglobulin molecules, such as kappa lightchain immunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term “control sequence” as used herein refers to polynucleotidesequences which are necessary to effect the expression and processing ofcoding sequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences. The term “polynucleotide” as referred to herein means apolymeric boron of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNA.

The term oligonucleotide referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g., for probes, although oligonucleotides may be double stranded,e.g., for use in the construction of a gene mutant. Oligonucleotides ofthe invention are either sense or antisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes Oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselerloate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoronmidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984),Stein et al. Nucl. Acids Res. 16:3209 (1988), Zon et al. Anti CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990). An oligonucleotidecan include a label for detection, if desired.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof in accordance with the invention selectively hybridize tonucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. High stringency conditions can be used to achieveselective hybridization conditions as known in the art and discussedherein. Generally, the nucleic acid sequence homology between thepolynucleotides, oligonucleotides, and fragments of the invention and anucleic acid sequence of interest will be at least 80%, and moretypically with preferably increasing homologies of at least 85%, 90%,95%, 99%, and 100%. Two amino acid sequences are homologous if there isa partial or complete identity between their sequences. For example, 85%homology means that 85% of the amino acids are identical when the twosequences are aligned for maximum matching. Gaps (in either of the twosequences being matched) are allowed in maximizing matching gap lengthsof 5 or less are preferred with 2 or less being more preferred.Alternatively and preferably, two protein sequences (or polypeptidesequences derived from them of at least 30 amino acids in length) arehomologous, as this term is used herein, if they have an alignment scoreof at more than 5 (in standard deviation units) using the program ALIGNwith the mutation data matrix and a gap penalty of 6 or greater. SeeDayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110(Volume 5, National Biomedical Research Foundation (1972)) andSupplement 2 to this volume, pp. 1-10. The two sequences or partsthereof are more preferably homologous if their amino acids are greaterthan or equal to 50% identical when optimally aligned using the ALIGNprogram. The term “corresponds to” is used herein to mean that apolynucleotide sequence is homologous (i.e., is identical, not strictlyevolutionarily related) to all or a portion of a referencepolynucleotide sequence, or that a polypeptide sequence is identical toa reference polypeptide sequence. In contradistinction, the term“complementary to” is used herein to mean that the complementarysequence is homologous to all or a portion of a reference polynucleotidesequence. For illustration, the nucleotide sequence “TATAC” correspondsto a reference sequence “TATAC” and is complementary to a referencesequence “GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence”, “comparison window”, “sequence identity”, “percentage ofsequence identity”, and “substantial identity”. A “reference sequence”is a defined sequence used as a basis for a sequence comparison areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window”, as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.)85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison,Wis.), Geneworks, or MacVector software packages), or by inspection, andthe best alignment (i.e., resulting in the highest percentage ofhomology over the comparison window) generated by the various methods isselected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland? Mass. (1991)). Stereoisomers (e.g., D-amino acids) of thetwenty conventional amino acids, unnatural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and otherunconventional amino acids may also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4 hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, the lefthanddirection is the amino terminal direction and the righthand direction isthe carboxy-terminal direction, in accordance with standard usage andconvention.

Similarly, unless specified otherwise, the lefthand end ofsingle-stranded polynucleotide sequences is the 5′ end the lefthanddirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”, sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity.

Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine valine,glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic amino acids are aspartate, glutamate; (2)basic amino acids are lysine, arginine, histidine; (3) non-polar aminoacids are alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan, and (4) uncharged polar amino acids are glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Thehydrophilic amino acids include arginine, asparagine, aspartate,glutamine, glutamate, histidine, lysine, serine, and threonine. Thehydrophobic amino acids include alanine, cysteine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan, tyrosine and valine.Other families of amino acids include (i) serine and threonine, whichare the aliphatic-hydroxy family; (ii) asparagine and glutamine, whichare the amide containing family; (iii) alanine, valine, leucine andisoleucine, which are the aliphatic family; and (iv) phenylalanine,tryptophan, and tyrosine, which are the aromatic family. For example, itis reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting molecule, especially if the replacement doesnot involve an amino acid within a framework site. Whether an amino acidchange results in a functional peptide can readily be determined byassaying the specific activity of the polypeptide derivative. Assays aredescribed in detail herein. Fragments or analogs of antibodies orimmunoglobulin molecules can be readily prepared by those of ordinaryskill in the art. Preferred amino- and carboxy-termini of fragments oranalogs occur near boundaries of functional domains. Structural andfunctional domains can be identified by comparison of the nucleotideand/or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure and/or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with theinvention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al. Nature 354:105 (1991).

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long' morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long. The term“analog” as used herein refers to polypeptides which are comprised of asegment of at least 25 amino acids that has substantial identity to aportion of a deduced amino acid sequence and which has at least one ofthe following properties: (1) specific binding to CD3, under suitablebinding conditions, (2) ability to block appropriate CD3 binding, or (3)ability to inhibit CD3-expressing cell growth in vitro or in vivo.Typically, polypeptide analogs comprise a conservative amino acidsubstitution (or addition or deletion) with respect to thenaturally-occurring sequence. Analogs typically are at least 20 aminoacids long, preferably at least 50 amino acids long or longer, and canoften be as long as a full-length naturally-occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drus with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986), Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987). Such compounds are often developed with theaid of computerized molecular modeling. Peptide mimetics that arestructurally similar to therapeutically useful peptides may be used toproduce an equivalent therapeutic or prophylactic effect. Generally,peptidomimetics are structurally similar to a paradigm polypeptide(i.e., a polypeptide that has a biochemical property or pharmacologicalactivity), such as human antibody, but have one or more peptide linkagesoptionally replaced by a linkage selected from the group consisting of:—CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH—(cis and trans), —COCH₂—, CH(OH)CH₂—,and —CH₂SO—, by methods well known in the art. Systematic substitutionof one or more amino acids of a consensus sequence with a D-amino acidof the same type (e.g., D-lysine in place of L-lysine) may be used togenerate more stable peptides. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation may be generated by methods known in the art (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992)); for example, by addinginternal cysteine residues capable of forming intramolecular disulfidebridges which cyclize the peptide.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I) fluorescent labels (e.g., FITC, rhodamine, lanthanidephosphors), enzymatic labels (e.g., horseradish peroxidase,p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance. The term “pharmaceutical agent ordrug” as used herein refers to a chemical compound or compositioncapable of inducing a desired therapeutic effect when properlyadministered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

The term “antineoplastic agent” is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, particularly a malignant (cancerous) lesion,such as a carcinoma, sarcoma, lymphoma, or leukemia. Inhibition ofmetastasis is frequently a property of antineoplastic agents.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present.

Generally, a substantially pure composition will comprise more thanabout 80 percent of all macromolecular species present in thecomposition, more preferably more than about 85%, 90%, 95%, and 99%.Most preferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The term patient includes human and veterinary subjects.

Human Antibodies and Humanization of Antibodies

A huCD3 antibody is generated, for example, by immunizing xenogenic micecapable of developing fully human antibodies (see Example 1). An IgGhuCD3 antibody is generated, for example, by converting an IgM anti-CD3antibody produced by a transgenic mouse (see Example 2). Alternatively,such a huCD3 antibody is developed, for example, using phase-displaymethods using antibodies containing only human sequences. Suchapproaches are well-known in the art, e.g., in WO92/01047 and U.S. Pat.No. 6,521,404, which are hereby incorporated by reference. In thisapproach, a combinatorial library of phage carrying random pairs oflight and heavy chains are screened using natural or recombinant sourceof CD3 or fragments thereof.

This invention includes methods to produce a huCD3 antibody by a processwherein at least one step of the process includes immunizing atransgenic, non-human animal with human CD3 protein. Some of theendogenous heavy and/or kappa light chain loci of this xenogenicnon-human animal have been disabled and are incapable of therearrangement required to generate genes encoding immunoglobulins inresponse to an antigen. In addition, at least one human heavy chainlocus and at least one human light chain locus have been stablytransfected into the animal. Thus, in response to an administeredantigen, the human loci rearrange to provide genes encoding humanvariable regions immunospecific for the antigen. Upon immunization,therefore, the xenomouse produces B-cells that secrete fully humanimmunoglobulins.

A variety of techniques are well-known in the art for producingxenogenic non-human animals. For example, see U.S. Pat. No. 6,075,181and No. 6,150,584. By one strategy, the xenogeneic (human) heavy andlight chain immunoglobulin genes are introduced into the host germ line(e.g., sperm or oocytes) and, in separate steps, the corresponding hostgenes are rendered non-functional by inactivation using homologousrecombination. Human heavy and light chain immunoglobulin genes arereconstructed in an appropriate eukaryotic or prokaryotic microorganism,and the resulting DNA fragments are introduced into the appropriatehost, for example, the pronuclei of fertilized mouse oocytes orembryonic stem cells. Inactivation of the endogenous host immunoglobulinloci is achieved by targeted disruption of the appropriate loci byhomologous recombination in the host cells, particularly embryonic stemcells or pronuclei of fertilized mouse oocytes. The targeted disruptioncan involve introduction of a lesion or deletion in the target locus, ordeletion within the target locus accompanied by insertion into thelocus, e.g., insertion of a selectable marker. In the case of embryonicstem cells, chimeric animals are generated which are derived in partfrom the modified embryonic stem cells and are capable of transmittingthe genetic modifications through the germ line. The mating of hostswith introduced human immunoglobulin loci to strains with inactivatedendogenous loci will yield animals whose antibody production is purelyxenogeneic, e.g., human.

In an alternative strategy, at least portions of the human heavy andlight chain immunoglobulin loci are used to replace directly thecorresponding endogenous immunoglobulin loci by homologous recombinationin embryonic stem cells. This results in simultaneous inactivation andreplacement of the endogenous immunoglobulin. This is followed by thegeneration of chimeric animals in which the embryonic stem cell-derivedcells can contribute to the germ lines.

For example, a B cell clone that expresses human anti-CD3 antibody isremoved from the xenogenic non-human animal and immortalized accordingto various methods known within the art. Such B cells may be deriveddirectly from the blood of the animal or from lymphoid tissues,including but not restricted to spleen, tonsils, lymph nodes, and bonemarrow. The resultant, immortalized B cells may be expanded and culturedin vitro to produce large, clinically applicable quantities of huCD3antibody. Alternatively, genes encoding the immunoglobulins with one ormore human variable regions can be recovered and expressed in adiffering cell type, including but not restricted to a mammalian cellculture system, in order to obtain the antibodies directly or individualchains thereof, composed of single chain F_(v) molecules.

In addition, the entire set of fully human anti-CD3 antibodies generatedby the xenogenic non-human animal may be screened to identify one suchclone with the optimal characteristics. Such characteristics include,for example, binding affinity to the human CD3 protein, stability of theinteraction as well as the isotype of the fully human anti-CD3 antibody.Clones from the entire set which have the desired characteristics thenare used as a source of nucleotide sequences encoding the desiredvariable regions, for further manipulation to generate antibodies withthese characteristics, in alternative cell systems, using conventionalrecombinant or transgenic techniques.

This general strategy was demonstrated in connection with generation ofthe first XenoMouse™ strains as published in 1994. See Green et al.Nature Genetics 7:13-21 (1994). This approach is further discussed anddelineated in U.S. patent application Ser. Nos. 07/466,008, filed Jan.12, 1990, 07/610,515, filed Nov. 8, 1990, 07/919,297, filed Jul. 24,1992, 07/922,649, filed Jul. 30, 1992, filed 08/031,801, filed Mar. 15,1993, 08/112,848, filed Aug. 27, 1993, 08/234,145, filed Apr. 28, 1994,08/376,279, filed Jan. 20, 1995, 08/430, 938, Apr. 27, 1995, 08/464,584,filed Jun. 5, 1995, 08/464,582, filed Jun. 5, 1995, 08/463,191, filedJun. 5, 1995, 08/462,837, filed Jun. 5, 1995, 08/486,853, filed Jun. 5,1995, 08/486,857, filed Jun. 5, 1995, 08/486,859, filed Jun. 5, 1995,08/462,513, filed Jun. 5, 1995, 08/724,752, filed Oct. 2, 1996, and08/759, 620, filed Dec. 3, 1996 and U.S. Pat. Nos. 6,162,963, 6,150,584,6,114,598, 6,075,181, and 5,939,598 and Japanese Patent Nos. 3 068 180B2, 3 068 506 B2, and 3 068 507 B2. See also Mendez et al. NatureGenetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med.:188:483-495 (1998). See also European Patent No., EP 0 463 151 B1, grantpublished Jun. 12, 1996, International Patent Application No., WO94/02602, published Feb. 3, 1994, International Patent Application No.,WO 96/34096, published Oct. 31, 1996, WO 98/24893, published Jun. 11,1998, WO 00/76310, published Dec. 21, 2000.

In an alternative approach, others have utilized a “minilocus” approach.In the minilocus approach, an exogenous Ig locus is mimicked through theinclusion of pieces (individual genes) from the Ig locus. Thus, one ormore VH genes, one or more D_(H) genes, one or more J_(H) genes, a muconstant region, and a second constant region (preferably a gammaconstant region) are formed into a construct for insertion into ananimal. This approach is described in U.S. Pat. No. 5,545,807 to Suraniet al. and U.S. Pat. Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425,5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299, and6,255,458 each to Lonberg and Kay, U.S. Pat. Nos. 5,591,669 and6,023.010 to Krimpenfort and Berns, U.S. Pat. Nos. 5,612,205, 5,721,367,and 5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi andDunn, and GenPharm International U.S. patent application Ser. Nos.07/574,748, filed Aug. 29, 1990, 07/575,962, filed Aug. 31, 1990,07/810,279, filed Dec. 17, 1991, 07/853,408, filed Mar. 18, 1992,07/904,068, filed Jun. 23, 1992, 07/990,860, filed Dec. 16, 1992,08/053,131, filed Apr. 26, 1993, 08/096,762, filed Jul. 22, 1993,08/155,301, filed Nov. 18, 1993, 08/161,739, filed Dec. 3, 1993,08/165,699, filed Dec. 10, 1993, 08/209,741, filed Mar. 9, 1994. Seealso European Patent No. 0 546 073 B1, International Patent ApplicationNos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884 andU.S. Pat. No. 5,981,175. See further Taylor et al., 1992, Chen et al.,1993, Tuaillon et al., 1993, Choi et al., 1993, Lonberg et al., (1994),Taylor et al., (1994), and Tuaillon et al., (1995), Fishwild et al.,(1996).

An advantage of the minilocus approach is the rapidity with whichconstructs including portions of the Ig locus can be generated andintroduced into animals. Commensurately, however, a significantdisadvantage of the minilocus approach is that, in theory, insufficientdiversity is introduced through the inclusion of small numbers of V, D,and J genes. Indeed, the published work appears to support this concern.B-cell development and antibody production of animals produced throughuse of the minilocus approach appear stunted. Therefore, researchsurrounding the present invention has consistently been directed towardsthe introduction of large portions of the Ig locus in order to achievegreater diversity and in an effort to reconstitute the immune repertoireof the animals.

Kirin has also demonstrated the generation of human antibodies from micein which, through microcell fusion, large pieces of chromosomes, orentire chromosomes, have been introduced. See European PatentApplication Nos. 773 288 and 843 961.

Human anti-mouse antibody (HAMA) responses have led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and a immune variable region, itis expected that certain human anti-chimeric antibody (HACA) responseswill be observed, particularly in chronic or multi-dose utilizations ofthe antibody. Thus, it would be desirable to provide fully humanantibodies against CD3 in order to vitiate concerns and/or effects ofHAMA or HACA response.

The production of antibodies with reduced immunogenicity is alsoaccomplished via humanization and display techniques using appropriatelibraries. It will be appreciated that murine antibodies or antibodiesfrom other species can be humanized or primatized using techniques wellknown in the art. See e.g., Winter and Harris Immunol Today 14:43 46(1993) and Wright et al. Crit, Reviews in Immunol. 12125-168 (1992). Theantibody of interest may be engineered by recombinant DNA techniques tosubstitute the CH1, CH2, CH3, hinge domains, and/or the framework domainwith the corresponding human sequence (See WO 92102190 and U.S. Pat.Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and5,777,085). Also, the use of Ig cDNA for construction of chimericimmunoglobulin genes is known in the art (Liu et al. P.N.A.S. 84:3439(1987) and J. Immunol. 139:3521 (1987)). mRNA is isolated from ahybridoma or other cell producing the antibody and used to produce cDNA.The cDNA of interest may be amplified by the polymerase chain reactionusing specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).Alternatively, a library is made and screened to isolate the sequence ofinterest. The DNA sequence encoding the variable region of the antibodyis then fused to human constant region sequences. The sequences of humanconstant regions genes may be found in Kabat et al. (1991) Sequences ofProteins of immunological Interest, N.I.H. publication no. 91-3242.Human C region genes are readily available from known clones. The choiceof isotype will be guided by the desired effector functions, such ascomplement fixation, or activity in antibody-dependent cellularcytotoxicity. Preferred isotypes are IgG1, IgG3 and IgG4. Either of thehuman light chain constant regions, kappa or lambda, may be used. Thechimeric, humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g., by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as priers to introduce useful restriction sitesinto the J region for subsequent linkage of V region segments to human Cregion segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL-31sequence can be easily inserted and expressed. In such vectors, splicingusually occurs between the splice donor site in the inserted J regionand the splice acceptor site preceding the human C region, and also atthe splice regions that occur within the human CH exons. Polyadenylationand transcription termination occur at native chromosomal sitesdownstream of the coding regions. The resulting chimeric antibody may bejoined to any strong promoter, including retroviral LTRs, e.g., SV-40early promoter, (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Roussarcoma virus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and moloneymurine leukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)). Also,as will be appreciated, native Ig promoters and the like may be used.

Further, human antibodies or antibodies from other species can begenerated through display type technologies, including, withoutlimitation, phage display, retroviral display, ribosomal display, andother techniques, using techniques well known in the art and theresulting molecules can be subjected to additional maturation, such asaffinity maturation, as such techniques are well known in the art.Wright and Harris, supra., Hanes and Plucthau PEAS USA 94:4937-4942(1997) (ribosomal display), Parmley and Smith Gene 73:305-318 (1988)(phage display), Scott TIB5 17:241-245 (1992), Cwirla et al. PNAS USA87:6378-6382 (1990), Russel et al. Nucl. Acids Research 21:1081-1085(1993), Hoganboom et al. Immunol. Reviews 130:43-68 (1992), Chiswell andMcCafferty TIBTECH; 10:80-8A (1992), and U.S. Pat. No. 5,733,743. Ifdisplay technologies are utilized to produce antibodies that are nothuman, such antibodies can be humanized as described above.

Using these techniques, antibodies can be generated to CD3 expressingcells, CD3 itself, forms of CD3, epitopes or peptides thereof, andexpression libraries thereto (See e.g., U.S. Pat. No. 5,703,057) whichcan thereafter be screened as described above for the activitiesdescribed above.

Design and Generation of Other Therapeutics

In accordance with the present invention and based on the activity ofthe antibodies that are produced and characterized herein with respectto CD3, the design of other therapeutic modalities beyond antibodymoieties is facilitated. Such modalities include, without limitation,advanced antibody therapeutics, such as bispecific antibodies,immunotoxins, and radiolabeled therapeutics, generation of peptidetherapeutics, gene therapies, particularly intrabodies, antisensetherapeutics, and small molecules.

For example, in connection with bispecific antibodies, bispecificantibodies can be generated that comprise (i) two antibodies one with aspecificity to CD3 and another to a second molecule that are conjugatedtogether, (ii) a single antibody that has one chain specific to CD3 anda second chain specific to a second molecule, or (iii) a single chainantibody that has specificity to CD3 and the other molecule. Suchbispecific antibodies can be generated using techniques that are wellknown for example, in connection with (i) and (ii) See e.g., Fanger etal. Immunol Methods 4:72-81 (1994) and Wright and Harris, supra, and inconnection with (iii) See e.g., Traunecker et al. Int. J. Cancer(Suppl.) 7:51-52 (1992). In each case, the second specificity can bemade to the heavy chain activation receptors, including, withoutlimitation, CD16 or CD64 (See e.g., Deo et al. 18:127 (1997)) or CD89(See e.g., Valerius et al. Blood 90:4485-4492 (1997)). Bispecificantibodies prepared in accordance with the foregoing would be likely tokill cells expressing CD3, and particularly those cells in which the CD3antibodies of the invention are effective.

In connection with immunotoxins, antibodies can be modified to act asimmunotoxins utilizing techniques that are well known in the art. Seee.g., Vitetta Immunol Today 14:252 (1993). See also U.S. Pat. No.5,194,594. In connection with the preparation of radiolabeledantibodies, such modified antibodies can also be readily preparedutilizing techniques that are well known in the art. See e.g., Junghanset al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition,Chafner and Longo, eds., Lippincott Raven (1996)). See also U.S. Pat.Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471,and 5,697,902. Each of immunotoxins and radiolabeled molecules would belikely to kill cells expressing CD3, and particularly those cells inwhich the antibodies of the invention are effective.

In connection with the generation of therapeutic peptides, through theutilization of structural information related to CD3 and antibodiesthereto, such as the antibodies of the invention or screening of peptidelibraries, therapeutic peptides can be generated that are directedagainst CD3. Design and screening of peptide therapeutics is discussedin connection with Houghten et al. Biotechniques 13:412-421 (1992),Houghten PNAS USA 82:5131-5135 (1985), Pinalla et al. Biotechniques13:901-905 (1992), Blake and Litzi-Davis BioConjugate Chem. 3:510-513(1992). Immunotoxins and radiolabeled molecules can also be prepared,and in a similar manner, in connection with peptidic moieties asdiscussed above in connection with antibodies. Assuming that the CD3molecule (or a form, such as a splice variant or alternate form) isfunctionally active in a disease process, it will also be possible todesign gene and antisense therapeutics thereto through conventionaltechniques. Such modalities can be utilized for modulating the functionof CD3. In connection therewith the antibodies of the present inventionfacilitate design and use of functional assays related thereto. A designand strategy for antisense therapeutics is discussed in detail inInternational Patent Application No. WO 94/29444. Design and strategiesfor gene therapy are well known. However, in particular, the use of genetherapeutic techniques involving intrabodies could prove to beparticularly advantageous. See e.g., Chen et al. Human Gene Therapy5:595-601 (1994) and Marasco Gene Therapy 4:11-15 (1997). General designof and considerations related to gene therapeutics is also discussed inInternational Patent Application No. WO 97/38137.

Knowledge gleaned from the structure of the CD3 molecule and itsinteractions with other molecules in accordance with the presentinvention, such as the antibodies of the invention, and others can beutilized to rationally design additional therapeutic modalities. In thisregard, rational drug design techniques such as X-ray crystallography,computer-aided (or assisted) molecular modeling (CAMM), quantitative orqualitative structure-activity relationship (QSAR), and similartechnologies can be utilized to focus drug discovery efforts. Rationaldesign allows prediction of protein or synthetic structures which caninteract with the molecule or specific forms thereof which can be usedto modify or modulate the activity of CD3. Such structures can besynthesized chemically or expressed in biological systems. This approachhas been reviewed in Capsey et al. Genetically Engineered HumanTherapeutic Drugs (Stockton Press, NY (1988)). Further, combinatoriallibraries can be designed and synthesized and used in screeningprograms, such as high throughput screening efforts.

Therapeutic Administration and Formulations

It will be appreciated that administration of therapeutic entities inaccordance with the invention will be administered with suitablecarriers, excipients, and other agents that are incorporated intoformulations to provide improved transfer, delivery, tolerance, and thelike. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, Pa.(1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration. See also Baldrick P. “Pharmaceutical excipientdevelopment: the need for preclinical guidance.” Regul. ToxicolPharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and developmentof solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000),Charman W N “Lipids, lipophilic drugs, and oral drug delivery-someemerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al.“Compendium of excipients for parenteral formulations” PDA J Pharm SciTechnol. 52:238-311 (1998) and the citations therein for additionalinformation related to formulations, excipients and carriers well knownto pharmaceutical chemists.

Therapeutic formulations of the invention, which include a huCD3antibody of the invention, are used to treat or alleviate a symptomassociated with an immune-related disorder, such as, for example, anautoimmune disease or an inflammatory disorder.

Autoimmune diseases include, for example, Acquired ImmunodeficiencySyndrome (AIDS, which is a viral disease with an autoimmune component),alopecia greata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune inner ear disease (AIED), autoimmunelymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura(ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitishepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS),chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricialpemphigold, cold agglutinin disease, crest syndrome, Crohn's disease,Degos' disease, dermatomyositis-juvenile, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease,Guillain-Barr syndrome, Hashimoto's thyroiditis, idiopathic pulmonaryfibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy,insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still'sdisease), juvenile rheumatoid arthritis, Ménière's disease, mixedconnective tissue disease, multiple sclerosis, myasthenia gravis,pernacious anemia, polyarteritis nodosa, polychondritis, polyglandularsyndromes, polymyalgia rheumatica, polymyositis and dermatomyositis,primary agammaglobulinemia, primary biliary cirrhosis, psoriasis,psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumaticfever, rheumatoid arthritis, sarcoidosis, scleroderma (progressivesystemic sclerosis (PSS), also known as systemic sclerosis (SS)),Sjögren's syndrome, stiff-man syndrome, systemic lupus erythematosus,Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerativecolitis, uveitis, vitiligo and Wegener's granulomatosis.

Inflammatory disorders, include, for example, chronic and acuteinflammatory disorders. Examples of inflammatory disorders includeAlzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis,bronchial asthma, eczema, glomerulonephritis, graft vs. host disease,hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation oftissue and organs, vasculitis, diabetic retinopathy and ventilatorinduced lung injury.

In one embodiment, the huCD3 antibody compositions of the invention areadministered in conjunction with a second agent such as, for example,GLP-1 or a beta cell resting compound (i.e., a compound that reduces orotherwise inhibits insulin release, such as potassium channel openers).Examples of suitable GLP-1 compounds are described in e.g., thepublished application U.S. 20040037826, and suitable beta cell restingcompounds are described in published application U.S. 20030235583, eachof which is hereby incorporated by reference in its entirety.

In another embodiment, the huCD3 antibody compositions used to treat animmune-related disorder are administered in combination with any of avariety of known anti-inflammatory and/or immunosuppressive compounds.Suitable known compounds include, but are not limited to methotrexate,cyclosporin A (including, for example, cyclosporin microemulsion),tacrolimus, corticosteroids, statins, interferon beta, Remicade(Infliximab), Enbrel (Etanercept) and Humira (Adalimumab).

For example, in the treatment of rheumatoid arthritis, the huCD3antibody compositions of the invention can be co-administered withcorticosteroids, methotrexate, cyclosporin A, statins, Remicade(Infliximab), Enbrel (Etanercept) and/or Humira (Adalimumab).

In the treatment of uveitis, the huCD3 antibody compositions can beadministered in conjunction with, e.g., corticosteroids, methotrexate,cyclosporin A, cyclophosphamide and/or statins. Likewise, patientsafflicted with a disease such as Crohn's Disease or psoriasis can betreated with a combination of a huCD3 antibody composition of theinvention and Remicaid (Infliximab), and/or Humira (Adalimumab).

Patients with multiple sclerosis can receive a combination of a huCD3antibody composition of the invention in combination with, e.g.,glatiramer acetate (Copaxone), interferon beta-1a (Avonex), interferonbeta-1a (Rebif), interferon beta-1b (Betaseron or Betaferon),mitoxantrone (Novantrone), dexamethasone (Decadron), methylprednisolone(Depo-Medrol), and/or prednisone (Deltasone) and/or statins.

The present invention also provides methods of treating or alleviating asymptom associated with an immune-related disorder or a symptomassociated with rejection following organ transplantation. For example,the compositions of the invention are used to treat or alleviate asymptom of any of the autoimmune diseases and inflammatory disordersdescribed herein.

The therapeutic compositions of the invention are also used asimmunosuppression agents in organ or tissue transplantation. As usedherein, “immunosuppression agent” refers to an agent whose action on theimmune system leads to the immediate or delayed reduction of theactivity of at least one pathway involved in an immune response, whetherthis response is naturally occurring or artificially triggered, whetherthis response takes place as part of the innate immune system, theadaptive immune system, or both. These immunosuppressive huCD3 antibodycompositions are administered to a subject prior to, during and/or afterorgan or tissue transplantation. For example, a huCD3 antibody of theinvention is used to treat or prevent rejection after organ or tissuetransplantation.

In one embodiment, the immunosuppressive huCD3 antibody compositions ofthe invention are administered in conjunction with a second agent suchas, for example, GLP-1 or a beta cell resting compound, as describedabove.

In another embodiment, these immunosuppressive huCD3 antibodycompositions are administered in combination with any of a variety ofknown anti-inflammatory and/or immunosuppressive compounds. Suitableanti-inflammatory and/or immunosuppressive compounds for use with thehuCD3 antibodies of the invention include, but are not limited to,methotrexate, cyclosporin A (including, for example, cyclosporinmicroemulsion), tacrolimus, corticosteroids and statins.

In yet another embodiment of the invention, a huCD3 antibody isadministered to a human individual upon detection of the presence ofauto-reactive antibodies within the human individual. Such auto-reactiveantibodies are known within the art as antibodies with binding affinityto one or more proteins expressed endogenously within the humanindividual. In one aspect of the invention, the human individual istested for the presence of auto-reactive antibodies specificallyinvolved in one or more autoimmune diseases as are well known within theart. In one specific embodiment, a human patient is tested for thepresence of antibodies against insulin, glutamic acid decarboxylaseand/or the IA-2 protein, and subsequently administered with a huCD3antibody upon positive detection of one or more such auto-reactiveantibodies.

In another embodiment of the invention, a huCD3 antibody is administeredinto human subjects to prevent, reduce or decrease the recruitment ofimmune cells into human tissues. A huCD3 antibody of the invention isadministered to a subject in need thereof to prevent and treatconditions associated with abnormal or deregulated immune cellrecruitment into tissue sites of human disease.

In another embodiment of the invention, a huCD3 antibody is administeredinto human subjects to prevent, reduce or decrease the extravasation anddiapedesis of immune cells into human tissues. Thus, the huCD3antibodies of the invention are administered to prevent and/or treatconditions associated with abnormal or deregulated immune cellinfiltration into tissue sites of human disease.

In another embodiment of the invention, a huCD3 antibody is administeredinto human subjects to prevent, reduce or decrease the effects mediatedby the release of cytokines within the human body. The term “cytokine”refers to all human cytokines known within the art that bindextracellular receptors upon the cell surface and thereby modulate cellfunction, including but not limited to IL-2, IFN-g, TNF-a, IL-4, IL-5,IL-6, IL-9, IL-10, and IL-13.

The release of cytokines can lead to a toxic condition known as thecytokine release syndrome (CRS), a common clinical complication thatoccurs, e.g., with the use of an anti-T cell antibody such as ATG(anti-thymocyte globulin) and OKT3 (a murine anti-human CD3 antibody).This syndrome is characterized by the excessive release of cytokinessuch as TNF, IFN-gamma and IL-2 into the circulation. The CRS occurs asa result of the simultaneous binding of the antibodies to CD3 (via thevariable region of the antibody) and the Fc Receptors and/or complementreceptors (via the constant region of the antibody) on other cells,thereby activating the T cells to release cytokines that produce asystemic inflammatory response characterized by hypotension, pyrexia andrigors. Symptoms of CRS include fever, chills, nausea, vomiting,hypotension, and dyspnea. Thus, a huCD3 antibody of the inventioncontains one or more mutations designed to prevent abnormal release andproduction of one or more cytokine(s) in vivo.

In another embodiment of the invention, a huCD3 antibody is administeredinto human subjects to prevent, reduce or decrease the effects mediatedby the release of cytokine receptors within the human body. The term“cytokine receptor” refers to all human cytokine receptors within theart that bind one or more cytokine(s), as defined herein, including butnot limited to receptors of the aforementioned cytokines. Thus, a huCD3antibody of the invention is administered to treat and/or preventconditions mediated through abnormal activation, binding or ligation ofone or more cytokine receptor(s) within the human body. It is furtherenvisioned that administration of the huCD3 antibody in vivo willdeplete the intracellular signaling mediated by cytokine receptor(s)within such human subject.

In one aspect of the invention, a huCD3 antibody is administered to ahuman individual upon decrease of pancreatic beta-cell function therein.In one embodiment, the individual is tested for beta-cell function,insulin secretion or c-peptide levels as are known within the art.Subsequently, upon notice of sufficient decrease of either theindicator, the human individual is administered with a sufficient dosageregimen of a huCD3 antibody to prevent further progression of autoimmunedestruction of beta-cell function therein.

Diagnostic and Prophylactic Formulations

The fully human anti-CD3 MAbs of the invention are used in diagnosticand prophylactic formulations. In one embodiment, a huCD3 MAb of theinvention is administered to patients that are at risk of developing oneof the aforementioned autoimmune diseases. A patient's predisposition toone or more of the aforementioned autoimmune diseases can be determinedusing genotypic, serological or biochemical markers. For example, thepresence of particular HLA subtypes and serological autoantibodies(against insulin, GAD65 and IA-2) are indicative of Type I diabetes.

In another embodiment of the invention, a huCD3 antibody is administeredto human individuals diagnosed with one or more of the aforementionedautoimmune diseases. Upon diagnosis, a huCD3 antibody is administered tomitigate or reverse the effects of autoimmunity. In one such example, ahuman individual diagnosed with Type I diabetes is administered withsufficient dose of a huCD3 antibody to restore pancreatic function andminimize damage of autoimmune infiltration into the pancreas. In anotherembodiment, a human individual diagnosed with rheumatoid arthritis isadministered with a huCD3 antibody to reduce immune cell infiltrationinto and destruction of limb joints.

Antibodies of the invention are also useful in the detection of CD3 inpatient samples and accordingly are useful as diagnostics. For example,the huCD3 antibodies of the invention are used in in vitro assays, e.g.,ELISA, to detect CD3 levels in a patient sample.

In one embodiment, a huCD3 antibody of the invention is immobilized on asolid support (e.g., the well(s) of a microtiter plate). The immobilizedantibody serves as a capture antibody for any CD3 that may be present ina test sample. Prior to contacting the immobilized antibody with apatient sample, the solid support is rinsed and treated with a blockingagent such as mink protein or albumin to prevent nonspecific adsorptionof the analyte.

Subsequently the wells were treated with a test sample suspected ofcontaining the antigen, or with a solution containing a standard amountof the antigen. Such a sample may be, e.g., a serum sample from asubject suspected of having levels of circulating antigen considered tobe diagnostic of a pathology. After rinsing away the test sample orstandard, the solid support is treated with a second antibody that isdetectably labeled. The labeled second antibody serves as a detectingantibody. The level of detectable label is measured, and theconcentration of CD3 antigen in the test sample is determined bycomparison with a standard curve developed from the standard samples.

It will be appreciated that based on the results obtained using thehuCD3 antibodies of the invention in an in vitro diagnostic assay, it ispossible to stage a disease (e.g., an autoimmune or inflammatorydisorder) in a subject based on expression levels of the CD3 antigen.For a given disease, samples of blood are taken from subjects diagnosedas being at various stages in the progression of the disease, and/or atvarious points in the therapeutic treatment of the disease. Using apopulation of samples that provides statistically significant resultsfor each stage of progression or therapy, a range of concentrations ofthe antigen that may be considered characteristic of each stage isdesignated.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the present invention.

Example 1 Generation of huCD3 Antibodies

Immunization Strategies: To generate a fully human huCD3 antibody, twolines of transgenic mice were utilized, the HuMab® mice and the KM™ mice(Medarex, Princeton N.J.). Initial immunization strategies followedwell-documented protocols from the literature for generating mouseantibodies. (See e.g., Kung P, et al., Science; 206(4416): 347-9 (1979);Kung P C, et al., Transplant Proc. (3 Suppl 1):141-6 (1980); Kung P C,et al., Int J Immunopharmacol. 3(3):175-81 (1981)). The standardprotocols known in the art failed to produce fully human anti-CD3antibodies in the HuMAb® or the KM Mouse™. For example, the followingimmunization strategies were unsuccessful and did not produce functionalantibodies in either the HuMAb® or KM Mice™:

-   -   immunization with thymocytes only or T-cells only    -   immunization with recombinant human CD3 material only    -   immunization with recombinant CD3 material in Freund's adjuvant    -   immunization with cell in Freund's adjuvant    -   immunization with thymocytes or T-cells co-administered with        soluble CD3    -   immunization with thymocytes or T-cells co-administered with        recombinant CD3-expressing cells

When these prior art immunization strategies are used in a BALB/c mouserather than a HuMAb® or KM™ mouse, these strategies produce a murineanti-CD3 antibody rather than a human anti-CD3 antibody.

Accordingly, novel immunization strategies were developed by varying thefollowing parameters:

-   -   types of immunogens employed    -   frequency of injection    -   types of adjuvants employed    -   types of co-stimulation techniques employed    -   routes of immunization employed    -   types of secondary lymphoid tissue used for fusion

A series of novel immunization strategies were developed, including forexample, (i) immunization with a viral particle expressing CD3 only, and(ii) immunization with co-stimulatory signals (e.g., CD40, CD27 orcombinations thereof) co-administered with T cells, thymocytes, or withcells that have been transfected to express recombinant CD3.

In a first novel immunization strategy, referred to herein as the“hyper-boost protocol”, a HuMAb® mouse (Medarex, Inc., Princeton, N.J.)or a KM™ mouse (Medarex, Inc., Kirin) was immunized by first injectinghuman cells, e.g., thymocytes or T-cells. At time points ranging from 1to 8 weeks after injection of the thymocytes and/or T-cells, the micereceived one or more subsequent “hyper-boost” injections. Thehyper-boost injection included, for example, soluble CD3 protein (e.g.,recombinant soluble CD3 protein), additional injections of thymocytes orT-cells, CD3-transfected cells, viral particles expressing high levelsof CD3, and combinations thereof. For example, the hyper-boost injectioncontained a combination of soluble CD3 protein and CD3-transfectedcells.

Preferably, in the hyper-boost immunization protocols, the immunizedmice received two final hyper-boost injections at −6 and −3 days priorto fusion of the lymph nodes and/or spleen. For example, in the KMMouse™, the fused tissue is derived from the spleen, and in the HuMAb®mouse, the fused tissue is derived from lymph nodes and/or splenictissue.

In one example of the hyper-boost immunization protocol, one HuMAb™mouse was immunized three times with human thymocytes (˜10⁶ cells) ondays 0, 7 and 28. The Chinese ovarian cells line (CHO) transfected withthe cDNA encoding human CD3 δ and ε chains (CHO/CD3, ˜10⁶ cells) wasthen injected on days 47 and 65. Another boost with a viral particleexpressing high levels of CD3δε at its surface was given on day 79.Finally, the mouse was injected with soluble recombinant human CD3δε onday 121 and 124 before fusion of the lymph nodes on day 127.

All immunizations were given subcutaneously with Ribi (Corixa Corp.,Seattle Wash.) as an adjuvant. A total of 8.5×10⁶ cells were fused. Onlyseven out of 470 hybridomas screened produced a fully human anti-CD3antibody, and all of the fully human anti-CD3 antibodies were IgMmolecules. Two of these anti-CD3 antibodies were selected as atherapeutic clinical candidates (FIGS. 1-3).

In a second example of the hyper-boost immunization protocol, one KM™mouse was immunized twice with soluble recombinant human CD3& on days 0and 25. Human thymocytes (˜10⁶ cells) were then used for boosting ondays 40, 49 and 56. The soluble recombinant CD3δε was injected twice ondays 70, 77, 84 and 91. The mouse T cell line transfected with the cDNAencoding human CD3 δ and ε chains (EL4/CD3, ˜10⁶ cells) was theninjected on day 98. Finally, the mouse was injected with solublerecombinant human CD3δε on day 101 before fusion of the spleen on day104. Immunizations were administered intraperitoneally with Alum as anadjuvant, except on day 70 where Ribi adjuvant was used. CpG was used asa costimulatory agent on day 0, 25, 84 and 91. A total of 1.27×10⁸ cellswere fused. Only five out of 743 hybridomas screened produced a fullyhuman anti-CD3 antibody, and all of antibodies produced were IgGmolecules. One of the fully human anti-CD3 antibodies was selected as atherapeutic clinical candidate (FIG. 4).

Selection Criteria: Therapeutic clinical candidates were selected usingthe following criteria. First, antibody-binding to CD3 positive cellsversus CD3 negative cells was analyzed. For this, Jurkat CD3 positivecells (J+) and Jurkat negative cells (J−) were incubated with thedifferent antibodies and binding was assessed by flow cytometry (FIG.9A). Second, a competition assay in which the ability of the candidateantibody to inhibit the binding of the murine anti-human OKT3 monoclonalantibody to CD3 positive cells was assessed using J+ cells andcompetition was assessed by flow cytometry (FIG. 9B). Next, antigenicmodulation of CD3 and the TCR from the surface of human peripheral bloodT cells was assessed by flow cytometry (FIG. 9C). Finally, a T-cellproliferation assay was performed using human peripheral blood T cellsdyed with CFSE and cell division was assessed by flow cytometry (FIG.9D).

Example 2 Isotype Switching of Human Anti-CD3 Antibodies

Some huCD3 antibodies produced using the novel protocols described inExample 1 were IgM antibodies. These IgM antibodies were “converted” toan IgG antibody, preferably to an IgG1 antibody. For example, the IgMantibodies were converted by a cloning procedure in which the VDJ regionof the gene encoding the IgM antibody was cloned into an IgG1 heavychain gene obtained from a vector that contains a gene encoding allotypeF gamma1. For conversion of the light chain, the IgM sequence was clonedinto a vector containing the kappa region. In Medarex mice, e.g., theHuMAb® mouse, multiple light chains are produced, due to a lack ofallelic exclusion.

Each combination of heavy and light chains was transfected into 293Tcells using the FuGENE 6 (Roche Diagnostics) transfection agentaccording to the manufacturer's guidelines. The secreted monoclonalantibodies were tested for optimized functionality, e.g., target antigenbinding, using the selection criteria described in Example 1.

Example 3 Antigenic Modulation Using the huCD3 Antibodies

The huCD3 antibodies of the invention are capable of antigenicmodulation, which is defined as the redistribution and elimination ofthe CD3-TCR complex induced by antibody binding. Cell surface expressionof other molecules on T cells, including, for example, CD4 is notaltered by exposure to an anti-CD3 antibody of the invention (FIG. 9C).

Example 4 Reducing the Toxic Cytokine Release Syndrome Generated byhuCD3 Antibodies

Preferably, the huCD3 antibodies of the invention include a mutation inthe Fc region, such that the mutation alters cytokine release syndrome.As described above, the cytokine release syndrome (CRS) is a commonimmediate complication that occurs with the use of an anti-T cellantibody such as ATG (anti-thymocyte globulin) and OKT3 (a murineanti-CD3 antibody). This syndrome is characterized by the excessiverelease of cytokines such as TNF, IFN-gamma and IL-2 into thecirculation. The cytokines released by the activated T cells produce atype of systemic inflammatory response similar to that found in severeinfection characterized by hypotension, pyrexia and rigors. Symptoms ofCRS include, for example, fever, chills, nausea, vomiting, hypotension,and dyspnea.

The huCD3 antibodies of the invention contain one or more mutations thatprevent heavy chain constant region-mediated release of one or morecytokine(s) in vivo. In one embodiment, the huCD3 antibodies of theinvention are IgG molecules having one or more of the followingmutations in a modified IgG γ1 backbone: “γ1 N297A”, in which theasparagine residue at position 297 is replaced with an alanine residue;“γ1 L234/A, L235/A”, in which the leucine residues at positions 234 and235 are replaced with alanine residues; “γ1 L234/A; L235/E”, in whichthe leucine residue at position 234 is replaced with an alanine residue,while the leucine residue at position 235 is replaced with a glutamicacid residue; “γ1 L235/E” in which the leucine residue at position 235is replaced with a glutamic acid residue; and “γ1 D265/A” in which theaspartic acid residue at position 265 is replaced with an alanineresidue. The numbering of the heavy chain residues described herein isthat of the EU index (see Kabat et al., “Proteins of ImmunologicalInterest”, US Dept. of Health & Human Services (1983)), as shown, e.g.,in U.S. Pat. Nos. 5,624,821 and 5,648,260, the contents of which arehereby incorporated in its entirety by reference.

Other IgG γ1 backbone modifications that can be used in the huCD3antibodies of the invention include, for example, “A330/S” in which thealanine residue at position 330 is replaced with a serine residue,and/or “P331/S” in which the proline residue at position 331 is replacedwith a serine residue.

The fully human CD3 antibodies of the invention having a L²³⁴ L²³⁵→A²³⁴E²³⁵ mutation in the Fc region have a unique function—elimination ofcytokine release in the presence of the huCD3 antibody. Prior studieshave actually taught away from the use of an L→E mutation (see e.g., Xuet al., Cellular Immunology, 200, pp. 16-26 (2000), at p. 23). However,these particular two mutations at positions 234 and 235 (i.e., L²³⁴L²³⁵→A²³⁴ E²³⁵) eliminated the cytokine release syndrome, as assessedperipheral human blood mononuclear cell in vitro assay system (FIGS.11A, 11B). In this assay, peripheral human blood mononuclear cells wereisolated using a ficoll gradient, labeled with CFSE and the CFSE-labeledcells were then plated into 96 well plates. The various monoclonalantibodies were added at various dilutions and incubated for 72 hours at37° C. After 6 hours, 50 μl of supernatant was removed to evaluate TNFrelease by ELISA. After 48 hours, 50 μl of supernatant was removed toevaluate IFN-γ release by ELISA. After 72 hours, the cells wereharvested and proliferation was assessed by FACS using CFSE-labelingintensity.

Thus, contrary to wild type heavy chains, and contrary to a series ofother mutations that had been described by others (e.g., TolerX(aglycosylation mutation), Bluestone (L²³⁴ L²³⁵→A²³⁴ A²³⁵ mutation) (seee.g., U.S. Pat. No. 5,885,573)), which all retain a significant level ofcytokine release effect, the L²³⁴ L²³⁵→A²³⁴ E²³⁵ mutations of the huCD3antibodies of the invention do not exhibit cytokine release phenomenon.The level of remaining cytokine release effect was 100% for the wildtype Fc, about 50 to 60% for the Bluestone (L²³⁴ L²³⁵→A²³⁴ A²³⁵)mutation and undetectable for Ala/Glu Fc mutations described herein(FIG. 11A, 11B).

Example 5 Peptide Array Identification of the huCD3 Antibody-BindingEpitope

The synthesis and ELISA screening of large numbers of peptides have beenused to determine the amino acid residues involved in the epitope forvarious monoclonal antibodies. (See e.g., Geysen et al., J ImmunolMethods, vol. 102(2):259-74 (1987)). In the experiments describedherein, arrays of overlapping peptides derived from the amino acidsequence of the CD3 epsilon chain were purchased from Jerini (Berlin,Germany) and subsequently tested for a pattern of binding by the fullyhuman anti-CD3 mAbs of the invention.

The peptides in the arrays were produced using the “SPOT synthesis”technique for direct chemical synthesis on membrane supports (see Frankand Overwin, Meth Mol Biol, vol. 66:149-169 (1996); Kramer andSchneider-Mergener, Meth Mol Biol, vol. 87:25-39 (1998)). The linear14-mer peptides were covalently bound to a Whatman 50 cellulose supportby the C-terminus, leaving the N-terminus free (i.e., unbound). Usingstandard western blotting techniques, these solid phase-bound peptidesrevealed that the 28F11 monoclonal recognized an overlapping set ofamino acids in proximity to the N terminus (FIG. 10).

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1. An isolated fully human monoclonal CD3 antibody or fragment thereof,wherein said antibody has the following characteristics: a. binds to CD3positive (CD3+) cells but not CD3 negative (CD3−) cells; b. modulatesthe cell surface expression level or activity of CD3; c. decreases thecell surface expression level or activity of the T cell receptor; and d.binds an epitope that wholly or partially includes the amino acidsequence EMGGITQTPYKVSISGT (SEQ ID NO:67).
 2. The antibody of claim 1,wherein said antibody inhibits binding of the murine anti-human OKT3monoclonal antibody to a T-lymphocyte.
 3. The antibody of claim 1,wherein said antibody competes for binding to CD3 with an antibodyselected from: (i) an anti-CD3 antibody having a variable heavy chainconsisting of the amino acid sequence of SEQ ID NO: 2 and a variablelight chain consisting of the amino acid sequence of SEQ ID NO: 4; (ii)an anti-CD3 antibody having a variable heavy chain consisting of theamino acid sequence of SEQ ID NO: 6 and a variable light chainconsisting of the amino acid sequence of SEQ ID NO: 8; (iii) an anti-CD3antibody having a variable heavy chain consisting of the amino acidsequence of SEQ ID NO: 10 and a variable light chain consisting of theamino acid sequence of SEQ ID NOs: 16, 17, 18, 19 or 20; and (iv) ananti-CD3 antibody having a variable heavy chain consisting of the aminoacid sequence of SEQ ID NO: 22 and a variable light chain consisting ofthe amino acid sequence of SEQ ID NOs: 25 or
 26. 4. The antibody ofclaim 1, wherein said antibody includes a mutation in the heavy chain atan amino acid residue at position 234, 235, 265, or 297 or combinationsthereof, and reduces the release of cytokines from a T-cell.
 5. Theantibody of claim 4, wherein said mutation results in an alanine orglutamic acid residue at said position.
 6. The antibody of claim 1,wherein said antibody is an IgG1 isotype and contains at least a firstmutation at position 234 and a second mutation at position 235, whereinsaid first mutation results in an alanine residue at position 234 andsaid second mutation results in a glutamic acid residue at position 235.7. An isolated fully human monoclonal antibody, wherein said antibodyhas a heavy chain with three complementarity determining regions (CDRs)comprising a VH CDR1 comprising the amino acid sequence SYGMH (SEQ IDNO:33); a VH CDR2 comprising an amino acid sequence selected from thegroup consisting of the amino acid sequences of IIWYDGSKKNYADSVKG (SEQID NO:34) and AIWYNGRKQDYADSVKG (SEQ ID NO:44); and a VH CDR3 comprisingthe amino acid sequence of GTGYNWFDP (SEQ ID NO:35); and a light chainwith three CDRs, comprising a VL CDR1 comprising an amino acid sequenceselected from the group consisting of the amino acid sequences ofRASQSVSSYLA (SEQ ID NO:30); RASQSVSSSYLA (SEQ ID NO:36); RASQGISSALA(SEQ ID NO:39) and WASQGISSYLA (SEQ ID NO:43); a VL CDR2 comprising anamino acid sequence selected from the group consisting of the amino acidsequences of DASNRAT (SEQ ID NO:31); GASSRAT (SEQ ID NO:37); YASSLQS(SEQ ID NO:40); DASSLGS (SEQ ID NO:42); and DASSLES (SEQ ID NO:46); anda VL CDR3 comprising an amino acid sequence selected from the groupconsisting of the amino acid sequences of QQYGSSPIT (SEQ ID NO:38);QQYYSTLT (SEQ ID NO:41); QQRSNWPWT (SEQ ID NO:45); and QQFNSYPIT (SEQ IDNO:47), wherein said antibody binds CD3.
 8. The antibody of claim 7,wherein said antibody further comprises a heavy chain variable aminoacid sequence selected from the group consisting of SEQ ID NOS: 10 and22, and a light chain variable amino acid sequence selected from thegroup consisting of SEQ ID NOS: 16-20, and
 26. 9. The antibody of claim7, wherein said antibody is an IgG1 isotype.
 10. An isolated fully humanmonoclonal antibody, wherein said antibody comprises a heavy chainvariable amino acid sequence selected from the group consisting of SEQID NOS: 10 and 22, and a light chain variable amino acid sequenceselected from the group consisting of SEQ ID NOS: 16-20, 25 and 26,wherein said antibody binds CD3.
 11. The antibody of claim 10, whereinsaid antibody is an IgG1 isotype.
 12. An isolated fully human monoclonalanti-CD3 antibody or fragment thereof, wherein said antibody comprises aV_(H) CDR1 region comprising the amino acid sequence YGMH (SEQ IDNO:58), a V_(H) CDR2 region comprising the amino acid sequence DSVKG(SEQ ID NO:59), and a V_(H) CDR3 region comprising the amino acidsequence X_(A)X_(B)GYX_(C)X_(D)FDX_(E) (SEQ ID NO:61).
 13. The antibodyof claim 12, wherein said V_(H) CDR2 region comprises the amino acidsequence IWYX₁GX₂X₃X₄X₅YX₆DSVKG (SEQ ID NO:60).
 14. The antibody ofclaim 14, wherein X_(i), X₂, X₃, X₄, X₅ and X₆ represent any amino acid.15. The antibody of claim 14, wherein X_(i), X₂, X₃ and X₄ arehydrophilic amino acids.
 16. The antibody of claim 14, wherein X_(i) isasparagine or aspartate, X₂ is arginine or serine, X₃ is lysine orasparagine, X₄ is lysine or glutamine, X₅ is aspartate, asparagine ortyrosine, and X₆ is valine or alanine.
 17. The antibody of claim 14,wherein said V_(H) CDR2 region comprises an amino acid sequence selectedfrom the group consisting of AIWYNGRKQDYADSVKG (SEQ ID NO:69),IIWYDGSKKNYADSVKG (SEQ ID NO:70), VIWYDGSKKYYVDSVKG (SEQ ID NO:71) andVIWYDGSNKYYADSVKG (SEQ ID NO:72).
 18. The antibody of claim 13, whereinX_(A), X_(B), X_(C), X_(D), and X_(E) represent any amino acid.
 19. Theantibody of claim 13, wherein X_(A) and X_(B) are neutral amino acids,X_(D) is an aromatic amino acid, and X_(E) is a hydrophobic amino acid.20. The antibody of claim 13, wherein X_(A) is glycine or glutamine,X_(B) is threonine or methionine, X_(C) is asparagine or tryptophan,X_(D) is tryptophan or histidine, and X_(E) is proline or leucine. 21.The antibody of claim 13, wherein said V_(H) CDR3 region comprises theamino acid sequence GTGYNWFDP (SEQ ID NO:62) or the amino acid sequenceQMGYWHFDL (SEQ ID NO:63).
 22. An isolated fully human monoclonalanti-CD3 antibody or fragment thereof, wherein said antibody comprises aframework 2 region (FWR2) comprising the amino acid sequenceWVRQAPGKGLEWV (SEQ ID NO:73), a framework 3 region (FRW3) comprising theamino acid sequence RFTISRDNSKNTLYLQMNSLRAEDTAVYYCA (SEQ ID NO:74), anda variable region located C-terminal to the CDR3 region, wherein saidvariable region comprises the amino acid sequence VTVSS (SEQ ID NO:64)at a position that is C-terminal to the CDR3 region.
 23. The antibody ofclaim 22, wherein said variable region located C-terminal to the CDR3region comprises the amino acid sequence GTLVTVSS (SEQ ID NO:65). 24.The antibody of claim 23, wherein said variable region locatedC-terminal to the CDR3 region comprises the amino acid sequenceWGRGTLVTVSS (SEQ ID NO:66).
 25. A method of alleviating a symptom of anautoimmune disease or inflammatory disorder, the method comprisingadministering an antibody of claim 1 to a subject in need thereof in anamount sufficient to alleviate the symptom of the autoimmune disease orinflammatory disorder in the subject.
 26. The method of claim 25,wherein said subject is a human.
 27. The method of claim 25, whereinsaid antibody has a heavy chain with three complementarity determiningregions (CDRs), comprising a VH CDR1 comprising an amino acid sequenceselected from the group consisting of the amino acid sequences of GYGMH(SEQ ID NO:27) and SYGMH (SEQ ID NO:33); a VH CDR2 comprising an aminoacid sequence selected from the group consisting of VIWYDGSKKYYVDSVKG(SEQ ID NO:28), IIWYDGSKKNYADSVKG (SEQ ID NO:34), and AIWYNGRKQDYADSVKG(SEQ ID NO:44); and a VH CDR3 comprising an amino acid sequence selectedfrom the group consisting of QMGYWHFDL (SEQ ID NO:29) and GTGYNWFDP (SEQID NO:35); and a light chain with three CDRs, comprising a VL CDR1comprising an amino acid sequence selected from the group consisting ofthe amino acid sequences of RASQSVSSYLA (SEQ ID NO:30), RASQSVSSSYLA(SEQ ID NO:36), RASQGISSALA (SEQ ID NO:39), and WASQGISSYLA (SEQ IDNO:43); a VL CDR2 comprising an amino acid sequence selected from thegroup consisting of DASNRAT (SEQ ID NO:31), GASSRAT (SEQ ID NO:37),YASSLQS (SEQ ID NO:40), DASSLGS (SEQ ID NO:42), and DASSLES (SEQ IDNO:46); and a VL CDR3 comprising an amino acid sequence selected fromthe group consisting of QQRSNWPPLT (SEQ ID NO:32); QQYGSSPIT (SEQ IDNO:38), QQYYSTLT (SEQ ID NO:41), QQRSNWPWT (SEQ ID NO:45), and QQFNSYPIT(SEQ ID NO:47).
 28. The method of claim 25, wherein said antibodycomprises a heavy chain variable amino acid sequence selected from thegroup consisting of SEQ ID NOS: 10 and 22 and a light chain variableamino acid sequence selected from the group consisting of SEQ ID NOS:16-20, 25 and
 26. 29. The method of claim 25, wherein said antibody isco-administered with an second agent selected from an anti-inflammatorycompound and an immunosuppressive compound.
 30. The method of claim 29,wherein the second agent is GLP-1, a beta cell resting compound,methotrexate, cyclosporin A, tacrolimus, corticosteroids, statins,interferon beta, glatiramer acetate, mitoxantrone, dexamethasone,methylprednisolone, prednisone, Infliximab, Etanercept or Adalimumab.